Inherited diseases such as SCID-X1, WAS, ADA, ALD and beta-thalassemia have been treated successfully using therapeutic gene transfer into hematopoietic stem cells. However, not all patients have benefited equally. There have been several adverse events in which integration of gene therapy vectors near cellular proto-oncogenes was associated with upregulation of transcription and leukemia, and in some subjects the gene therapy did not effectively ameliorate the underlying condition. We have carried out extensive studies of cell populations during human gene correction using vector integration site distribution data to infer the behavior of progenitor cells. We developed novel deep sequencing and bioinformatic methods for this purpose, yielding a wealth of data on successful and unsuccessful outcomes. Using these data and ongoing long term monitoring, we can address a new set of questions on the population biology of transduced cells, both to understand more fully the optimal strategies for human gene correction, and to understand development of the human hematopoietic system itself. We propose to acquire and analyze data on integration site distributions during long term patient monitoring. To expand our picture of blood cell development, we will also acquire and analyze data on VDJ recombination products in T-cells. In our first Aim, we will address the following hypotheses: 1) vectors with intact LTRs (including transcriptional enhancers) are much more active in driving expansion of cell clones than vectors without them, but some types of events with all vectors are consistent with insertional activation and vector driving~ 2) long term progenitor function differs depending on disease state, vector type, conditioning regimen and clinical condition of the subject~ 3) most transduced progenitor cells are in fact committed not just to the lymphoid or myeloid lineages, but even more specifically within each~ 4) progenitors are contributing cells to the periphery episodically, and this is further magnified by proliferation of antigen responsive cells and 5) stem cell activity wanes over time, but only very slowly. In our second Aim, which adds in TCR data, we will investigate the following further hypotheses: 1) TCR-beta repertoire sizes after SCID-X1 or WAS gene correction approach those observed in healthy adults~ 2) CD4+ and CD8+ T-cell repertoires contain mostly different TCR-beta recombination products, indicating mostly independent recombination and commitment~ 3) the number of cell divisions can be estimated from these data that link progenitors and mature T-cells~ 4) proliferation in response to antigen involves only a specific subset of progenitors and TCRs~ 5) TCR sequencing can be a sensitive marker for clonal expansion in leukemia and successful eradication of the leukemic clone by chemotherapy. Thus the proposed study will provide unique data useful both in optimizing gene therapy methods and understanding human hematopoiesis, and also provide a mechanism for supporting FDA-mandated long-term follow up of gene-corrected subjects.